title: A structural model of PpoA derived from SAXS-analysis-implications for substrate conversion.
abstract: In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement.
authors.affiliation: Georg-August-University, Albrecht-von-Haller-Institute, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Koch
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Tria
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Fielding
authors.forename: Florian
authors.initials: F
authors.lastname: Brodhun
authors.forename: Oliver
authors.initials: O
authors.lastname: Valerius
authors.forename: Kirstin
authors.initials: K
authors.lastname: Feussner
authors.forename: Gerhard H
authors.initials: GH
authors.lastname: Braus
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Marina
authors.initials: M
authors.lastname: Bennati
authors.forename: Ivo
authors.initials: I
authors.lastname: Feussner
mesh_terms: D001233:Aspergillus nidulans / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism*; D004583:Electrons; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D003577:Cytochrome P-450 Enzyme System; D049308:Dioxygenases
keywords: AOS; Cytochrome P450; D(max); DEER; DES; DOX; DiHODE; DiHOME; Dioxygenase; Double electron–electron resonance; EPR; Et(2)O; H(P)ODE; HPL; KODE; KOME; LOX; MALDI-TOF; MW; Oxylipin; PGHS; PGIS; Ppo; Psi-factor producing oxygenase; R(g); SAXS; Small-angle X-ray scattering; TXAS; V(p); allene oxide synthase; diethyl ether; dihydroxy linoleic acid; dihydroxy oleic acid; dioxygenase; divinyl ether synthase; double electron–electron resonance; electron paramagnetic resonance; hydro(pero)xy linoleic acid; hydroperoxide lyase; lipoxygenase; matrix-assisted laser desorption/ionization-time of flight; maximal particle dimension; molecular weight; oxo-linoleic acid; oxo-oleic acid; particle volume; precocious sexual inducer; prostacyclin synthase; prostaglandin endoperoxide synthase; psi; psi-factor producing oxygenase; radius of gyration; small-angle X-ray scattering; thromboxane synthase